Activation of resting T lymphocytes through the T cell antigen receptor (TCR) and its co-receptors is initiated by rapid but transient phosphorylation of several regulatory proteins or enzymes on tyrosine residues. At least six protein tyrosine kinases (PTKs) belonging to the Src, Syk, Csk and Tec families are known to be involved in the earliest events induced by these receptors. In contrast, only one protein tyrosine phosphatase (PTPase), CD45, has so far been found to be important in these early signaling events. It is clear than many more PTPases must be involved in a critical manner both in maintaining the resting state of the signaling machinery and in the active transmission of signals. This proposal focuses on the PTPases. Specific Aim 1 attempts to elucidate which known or novel PTPases are involved in the regulation of the initial/early events of TCR/CD3 plus CD28-induced T cell activation. First, we will focus on a number of early parameters of TCR and/or CD28 signaling, which can be measured by a number of rapid co- transfection/reporter assays we have developed. Positive findings will be analyzed in more details using stably transfected cell lines. The PTPases judged to be involved in signaling as result of this work, will be further analyzed in Specific Aim 2, which addresses the connection between the receptors and the PTPases to elucidate how they are recruited or used by the TCR-and/or CD28-induced pathways. The biological relevance of findings will be evaluated by adding phosphorylation sites, and testing these as in Sp. Aim 1. Specific Aim 3 examines the events downstream of the PTPases to determine what the PTPases do in signaling. We will attempt to identify these targets, with particular emphasis on the receptor subunits, the PTKs and selected signaling molecules. The experiments will identify the sites and effects of dephosphorylation. It is anticipated that they participate in signaling by dephosphorylating components of the signaling machinery either in a "housekeeping" manner or in a more regulated and specific manner. We will also investigate the possibility that PTPases participate in signaling independently of their catalytic activities, e.g. by acting as adapters or bridging molecules. We anticipate that the results obtained in this study will enable use to better understand the molecular events that follow TCR plus CD28 triggering and govern the onset of T cell proliferation. This will not only benefit our understanding of the initiation of an immune response, but also the normal regulation and proliferation in other cell types as well. This information, in turn, is crucial for the study of deregulated and malignant proliferation of cells e.g. in T cell lymphomas and other malignant diseases.